1. Field of the Invention
The present invention relates to a monoclonal antibody or fragment thereof, which binds specifically and/or selectively to human receptors of the Frizzled (FZD) family. It further relates to a hybridoma cell, which produces the antibody according to the invention. A further object of the present invention is a method for the detection and/or isolation of receptors of the FZD family or homologs or fragments thereof in a biological sample and a method for the identification and/or isolation of biological material that has or expresses receptors of the FZD family or homologs or fragments thereof. The invention further relates to the use of the antibody according to the invention or fragments thereof for the specific and/or selective detection and/or isolation of receptors of the FZD family, a composition comprising the monoclonal antibody according to the invention or fragments thereof, and a kit that comprises the monoclonal antibody according to the invention or fragments thereof.
2. Related Prior Art
Frizzled (FZD) receptors are proteins of the 7-transmembrane type, which have a sequence motif with seven α-helical domains, by means of which anchoring in the membrane takes place. This motif is typical for the so-called G protein-coupled receptors (GPCRs). Many authors therefore also include the Frizzled receptors in the GPCR superfamily; cf. for example Malbon C. C. (2004), “Frizzleds: new members of the superfamily of G-protein-coupled receptors”, Front. Biosci., Vol. 9, pages 1048 to 1058. Furthermore, the Frizzled receptors have a cysteine-rich domain (CRD) at their amino-terminal end.
Frizzled receptors are important components of the so-called Wnt (wingless-type) signal pathway, which is conserved both in vertebrates and in invertebrates. The most important ligands of the Frizzled receptors are the Wnt proteins, which induce intracellular events by binding to the extracellular portion of the Frizzled receptor. In addition to the FZD receptors, members of another class of cell surface receptors from the family of LDL-receptor-related proteins (LRP) are involved in the reception and transmission of Wnt signals.
Intracellular signal transduction can essentially take place by two different pathways. Ligands of the Wnt5A class stimulate signal transduction via the pertussis-toxin-sensitive group of heterotrimeric G proteins through the increase in the intracellular Ca2+ concentration and via the RhoA/c-jun-N-terminal kinase signal cascade (called the Wnt-Ca2+ pathway). With ligands of the Wnt1 class, in contrast, signal transduction takes place via another important intracellular signal protein, β-catenin (called the Wnt/β-catenin pathway). β-Catenin is a transcription activator. In the absence of a Wnt signal, a portion of the intracellular β-catenin is bound to the cytosolic tail of the so-called cadherin protein and another portion of the cytosolic β-catenin is located in a complex of the proteins axin, APC (adenomatous polyposis coli) and GSK-3β (glycogen synthase kinase-3). In this so-called degradation complex, β-catenin is phosphorylated by GSK-3β and so triggers its own ubiquitinylation and therefore degradation in proteasomes. Accordingly, in the absence of Wnt, β-catenin is unstable and cannot perform its function as a transcription activator. In the absence of Wnt, therefore, various Wnt-regulated genes are inactivated, which is ensured by a corepressor protein with the designation Groucho, which together with the transcription-regulating proteins LEF-1/TCF is bound to the DNA in the promoter region of these genes.
When Wnt binds to the Frizzled receptor and its receptor LRP, through an as yet unknown mechanism there is activation of the intracellular protein Dishevelled. Activated Dishevelled induces, again by an as yet unknown mechanism, the inactivation of GSK-3β in the degradation complex. As a result there is inhibition of the phosphorylation and degradation of β-catenin. β-Catenin is then stable and accumulates in the cytoplasm and in the nucleus. In the nucleus, β-catenin binds to LEF-1/TCF (lymphoid enhancer factor-1/T cell-specific factor), releases the Groucho factor and thus stimulates the transcription of Wnt-regulated genes. A review of the Wnt signal pathway is given for example in Huelsken J. and Behrens J. (2002), “The Wnt signalling pathway”, J. Cell Sci., Vol. 115, pages 3977 to 3978.
In addition to Wnt, another ligand for Frizzled receptors was found recently, which has the name Norrin; cf. Niehrs C. (2004), “Norrin and Frizzled: A New Vein for the Eye”, Developmental Cell, Vol. 6, pages 1 to 20.
Frizzled receptors play an important role in the differentiation and organogenesis of embryonic tissue, in oogenesis and development of the germinal layer and in the autoregeneration of stem cells. Consequently, a great many teams from the area of basic research are involved in the detailed characterization of these receptors.
However, Frizzled receptors also seem to play an important role in many diseases. Thus, these receptors have a key function in carcinogenesis; cf. Holcombe R. F. et al. (2002), “Expression of Wnt ligands and Frizzled receptors in colonic mucosa and in colon carcinoma”, J. Clin. Pathol., Vol. 55(4), pages 220 to 226, and Karim R. et al. (2004), “The significance of the Wnt pathway in the pathology of human cancers”, Pathology 36(2), pages 120 to 128.
In addition, a link was found with Norrie disease, an X-chromosomal-recessive disease characterized by degenerative changes in the retina; cf. Xu et al. (2004), “Vascular development in the retina and inner ear: control by Norrin and Frizzled4, a high-affinity ligand-receptor pair”, Cell, Vol. 116(6), pages 883 to 895; involvement of Frizzled receptors in the clinical picture of familial exudative vitreoretinopathy, a progressive disease of the retina; cf. Robitaille et al. (2002), “Mutant frizzled-4 disrupts retinal angiogenesis in familial exudative vitreoretinopathy”, Nat. Genet., Vol. 32(2), pages 326 to 330.
Moreover, Frizzled receptors are involved in the clinical picture of rheumatoid arthritis; cf. Sen et al. (2001), “Blockade of Wnt-5A/frizzled 5 signaling inhibits rheumatoid synoviocyte activation”, Arthritis Rheum., Vol. 44(4), pages 772 to 781. Furthermore, Frizzled receptors are described in the literature in connection with a great many other clinical pictures.
Against this background, both for research and for diagnostics and pharmaceutics, there is a great need for antibodies that are able to selectively recognize and/or identify Frizzled receptors.
Polyclonal antibodies against various Frizzled receptors are widely described in the state of the art, for example against human FZD-5 (Sen et al., loc. cit.), against mouse FZD-9 (Van Raay et al. (2001), “Frizzled 9 is expressed in neural precursor cells in the developing neural tube”, Dev. Genes Evol., Vol. 211(8-9), pages 453 to 457), or against human FZD-1 and FZD-2 (Holcombe R. F. et al., loc. cit.).
Polyclonal antibodies of this kind have the disadvantage that they are not of unlimited availability, and they can no longer be reproduced identically after use. Moreover, polyclonal antibodies naturally comprise a mixture of different antibodies, each directed against different antigenic structures of the particular epitope. Consequently it is not possible, by means of polyclonal antibodies, to identify the same antigenic structure reproducibly again and again.
Another disadvantage of polyclonal antibodies is that they sometimes display very high cross reactivities with other proteins that are not of interest, which greatly restricts their use in immunoblotting or in immunoprecipitation and makes a diagnostic or therapeutic use of this mixture impossible.
There is therefore an increased need for the supply of monoclonal antibodies that are directed against Frizzled receptors, and especially against the human variants. However, it turns out that production of these monoclonal antibodies is particularly difficult, as Frizzled receptors in the animal kingdom are very highly conserved with respect to their amino acid sequence and therefore their three-dimensional structure. It is therefore assumed that it is almost impossible to produce monoclonal antibodies by means of which a species-specific demarcation of the Frizzled receptors is possible.
The company R&D Systems Inc., Minneapolis, Minn., USA, offers monoclonal antibodies against the mouse variant of Frizzled-1 (Clone 162531), -3 (Clone 169310), -4 (Clone 145901) and -7 (Clone 151143). The manufacturer advertises that the respective human variants of the Frizzled receptors could also be detected with these antibodies. However, experts have shown, and the inventors have also verified, that satisfactory detection of the human variants is not possible by means of these antibodies. Rather, binding of these antibodies to human receptors of the Frizzled family is nonspecific and unselective, so that only a certain level of cross reactivity with the human variants combined with high affinity for other unwanted structures can be determined. Therefore it is not possible to use these monoclonal antibodies for selective and/or specific identification of human Frizzled receptors.